title: Cross-linking of pyruvate decarboxylase. Characterization of the native and substrate-activated enzyme states.
abstract: In order to demonstrate the role of the protein component of pyruvate decarboxylase in the mechanism of substrate activation, we have isolated and characterized two states of the enzyme, the non-activated and the substrate-activated state, by covalent linking with bifunctional reagents. Because of the fact that modification of the reactive amino groups by 2,4,6-trinitobenzenesulfonic acid or methyl propionimidate influences neither the catalytic nor the regulatory properties of pyruvate decarboxylase, we used bisimidates of different chain length in the modification experiments. Both the non-activated and the substrate-activated enzyme states could be characterized separately. The lag phase of product formation as a typical property of the native enzyme disappeared completely when the enzyme had been cross-linked in the presence of the substrate. The permanently activated enzyme state shows 85% of the activity of native pyruvate decarboxylase and is exclusively stabilized by intra-subunit links. Elimination and subsequent reincorporation of the cofactors thiamine pyrophosphate and magnesium ions resulted in a complete regaining of the properties of the permanently activated enzyme form. An inactive enzyme form was obtained after cross-linking of non-activated pyruvate decarboxylase at low ionic strength (less than 0.01). Using a disulfide-containing linker we could prove that the inactivity of the obtained enzyme preparation was only the result of the incorporated cross-links and not that of denaturation.
authors.affiliation: Biotechnikum, Martin-Luther-Universität Halle-Wittenberg, GDR.
authors.forename: S
authors.initials: S
authors.lastname: König
authors.forename: G
authors.initials: G
authors.lastname: Hübner
authors.forename: A
authors.initials: A
authors.lastname: Schellenberger
mesh_terms: D003432:Cross-Linking Reagents; D004789:Enzyme Activation; D007700:Kinetics; D015394:Molecular Structure; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D013438:Sulfhydryl Compounds; D011767:Pyruvate Decarboxylase